Meanings of the abbreviations used in the present description are as follows.                HA: Hyaluronic acid        BSA: Bovine serum albumin        CS-C: Chondroitin sulfate C        HA-BSA: HA-BSA conjugate        HABP: HA-binding protein        Biotin-HABP: Biotin labeled hyaluronic acid-binding protein        HRP-avidin: Horse-radish peroxidase labeled streptoavidin        OPD tablet: o-phenylenediamine tablet        ABTS tablet: 2,2′-azinobis(3-ethylbenzothiazoline-6-O-sulfonate)        TMB (+): N, N, N, N-tetramethylbenzidine (+)        TMB BLUE: N, N, N, N-tetramethylbenzidine blue        PBS: Salt-added sodium phosphate buffered saline        GPC: Gel permeation chromatography        SDS: Sodium dodecyl sulfate        TCA: Trichloroacetic acid        EDTA: Ethylenediamine tetraacetate        EGTA: Ethylene glycol-bis(2-aminoethylether)-N, N, N′,N′-tetraacetic acid        TLCK: Nα-p-Tosyl-lysine chloromethyl ketone        TPCK: N-p-Tosyl-phenylalanine chloromethyl ketone        
HA is a naturally-occurring unbranched polysaccharide composed of repeating glucuronic acid and N-acetyl glucosamine disaccharide units. It has recently been pointed out, for example, that HA molecules which have different molecular weights show different bioactivities and that the molecular weight of HA of biological samples may be changed in some of the diseases. Accordingly, the method of measuring the molecular weight of HA is important in the fields of pharmaceuticals, medical care, and so on.
The measurement of the molecular weight of HA has generally been performed with a limiting viscosity measurement and a method utilizing GPC. The former is based on the principle that HA with higher molecular weight shows higher viscosity, and the latter is based on the principle of the size-exclusion chromatography in which HA with higher molecular weight migrate through the column more rapidly.
However, the limiting viscosity method requires about 1 mg or more of HA in order to carry out the measurement. Further, even if the general automatic devices is used, the maximum number of samples that can be measured simultaneously is 5 or less, and it required time-consuming manipulation such as drying of a viscosity tube for each measurement cycle. In addition, HA-specific measurement can not be achieved if other glycosaminoglycans coexist, because they exhibit similar viscosities. Further, coexisting substances other than the glycosaminoglycans also affect a solution viscosity. And finally, the measurement can not be performed unless an aqueous solution of the purified samples.
Further, the GPC method requires about 10 μg or more of HA in order to carry out the measurement. Further, it required at least 1 hour to equilibrate the measurement system, and it also required the measurement of a series of the molecular weight standards for each analysis. Not less than 5 different standards are generally used and need to be measured per one analytical batch in principle. In addition, the time required for one measurement cycle is at least 30 minutes. So it required 4 hours or more for only 1 test sample and 9 hours or more for 10 samples. And the GPC of the HA, UV detection is most sensitive currently, however, it also detect other glycosaminoglycans. Therefore, if other glycosaminoglycans coexist, the chromatogram of the glycosaminoglycans overlaps the chromatogram of HA, and HA-specific measurement can not be achieved. Similarly, if other contaminants which have some UV absorption coexist, HA-specific measurement can not be achieved. In many biological samples, there exist proteins, nucleic acids, lipids. which are having a double bond, and exhibits ultra violet absorption. Thus, in the case where the method is used to measure HA in biological samples, It required some pretreatment for removal of those contaminants.
As described above, the methods of measuring a molecular weight of HA that has been used up to the present requires at least about 10 μg to 1 mg of samples, much time, and complex manipulations, and besides the number of samples that can be measured simultaneously is restricted.
On the other hand, there have been proposed methods for the measurement of HA utilizing a protein binding to HA (Patent Documents 1 and 2), both of which are methods for quantitative measurement. There has not been known a method for direct measurement of a molecular weight at all.                Patent Document 1: JP 06-41952 B        Patent Document 2: JP 2698563 B        